In this new proposal, we investigate our novel finding that a unique combination of hematopoietic stem cell cytokines (i.e. stem cell factor-SCF;interleukin 3- IL-3;and stromal-derived factor-1 alpha- SDF-11) control human endothelial cell (EC) tube morphogenesis and EC-pericyte tube co assembly under defined serum-free conditions (and in the absence of phorbol ester) in 3D collagen matrices. Interestingly, VEGF has no influence by itself and requires the synergistic action of these hematopoietic factors in order to exert an effect on EC sprouting, its major morphogenic influence. We propose the hypothesis that SCF, IL-3 and SDF-11 are critical regulators of vascular morphogenesis which act in a synergistic manner and are necessary cofactors for the action of known mediators of developmental vascularization (e.g. VEGF and BMP-4). Combined administration of c-Kit (the SCF receptor) and CXCR4 (the SDF-11 receptor) inhibitors or blocking antibodies to SCF and IL-3 leads to vascular hemorrhage phenotypes in developing quail embryos that relates to defects in vessel formation and remodeling. Also, we show that the three factors synergize to activate Src, B-Raf and Erk kinases and induce the expression of the 1221 integrin and Pak2 which are known regulators of vascular morphogenesis. We will elucidate the molecular basis for the signaling synergism of the three cytokines in ECs, how these factors interface with VEGF, BMP-4, and pericytes to control EC sprouting and how they affect both ECs and pericytes during tube co assembly and maturation events in vitro and in vivo. We propose three specific aims which are;Specific Aim #1. To elucidate the signaling mechanisms by which SCF, IL-3 and SDF-11 synergize to stimulate vasculogenic EC tube assembly in 3D extracellular matrices. Specific Aim #2. To determine how SCF, IL-3 and SDF-11 affect VEGF and BMP-4 signaling to control EC sprouting responses in 3D extracellular matrices. Specific Aim #3. To determine how SCF, IL-3 and SDF-11 act to regulate pericyte-induced EC sprouting responses and vasculogenic EC-pericyte tube co assembly in 3D extracellular matrices. PUBLIC HEALTH RELEVANCE: This work focuses on the ability of factors which are known to support the growth of immature blood cells to also be able to support the formation of blood vessels which carry blood cells. These factors bind to endothelial cells, which line blood vessels and to pericytes, a supporting cell that helps blood vessels form and become stable. A basic understanding of the mechanisms underlying how blood vessels form and become stabilized is critical in efforts to stimulate or inhibit the process in the context of various human diseases such as cardiovascular disease, diabetes or cancer.